BBB-conquering antibodies

Roche
has developed a new transferrin receptor-targeting strategy that dramatically increased delivery of
an antibody to the brain.1 The key advance was slimming the
technology, dubbed Brain Shuttle, down to a single transferrin receptor-binding
antibody fragment. The pharma showed proof of concept in mice by delivering an
anti-b-amyloid
mAb and now needs to adapt the system for primate brains.

The blood brain barrier (BBB) is composed of endothelial cells
that allow the passive exchange of smaller, lipophilic molecules between blood
and the brain parenchyma but are impermeable to larger, more hydrophobic
molecules such as many biologics.

BBB endothelial cells have evolved dedicated,
receptor-mediated transcytosis pathways to shuttle larger endogenous molecules
such as transferrin and insulin.2 Because transferrin is one of the few
macromolecules able to trek across the BBB, researchers have developed Trojan
horse delivery systems that harness transferrin receptor (TfR)-mediated
transcytosis to shuttle biologics into the brain.

First-generation Trojan horses typically consisted of an
entire TfR-binding antibody fused to a payload-usually a second antibody
against a disease-related target. However, the high affinity of the antibody
for TfR led to insufficient target engagement in the brains of animals.3

In 2011, Roche's Genentech Inc.
unit took steps toward addressing both drawbacks by lowering the affinity of a
TfR-directed antibody. The result was improved brain uptake.

Genentech then eliminated the need to fuse two antibodies
together by developing a bispecific, chimeric antibody. One antigen-binding
site was specific for TfR and the other for b-site APP-cleaving enzyme 1
(BACE1), an enzyme that
contributes to processing the amyloid precursor protein (APP) into toxic,
amyloidogenic forms.

The bispecific antibody achieved brain concentrations of
about 20 nM, which was up to 10-fold more than a monospecific, BACE1-directed
antibody, and reduced b-amyloid
(Ab) levels in a mouse model of Alzheimer's
disease (AD).4

Now, the Roche team, led by Per-Ola Freskgård and Anirvan
Ghosh, has taken a different approach. The group took a therapeutic antibody
called gantenerumab and fused a TfR-specific antibody
fragment to one (monovalent) or both (bivalent) of gantenerumab's heavy chains.

Freskgård is vice director and senior leader for
neuroscience at the Roche Pharma Research and Early
Development (pRED) section, and Ghosh is head of neuroscience discovery at
pRED. Gantenerumab, a HuCAL (Human
Combinatorial Antibody Library)-derived mAb targeting Ab, is partnered with MorphoSys AG
and is in Phase II/III testing to treat AD.

The monovalent version produced the better results-it was
efficiently transcytosed via TfRs across the BBB.

In a mouse model of AD, i.v. injections of the monovalent,
TfR-binding antibody resulted in 50-fold increased target engagement in amyloid
plaques in the brain compared with gantenerumab. The monovalent antibody
markedly reduced plaque burden in the animals' cortex and hippocampal brain
regions.

The findings were published in Neuron.

"The most important finding is that endothelial cells
at the BBB differentially process internalized TfR in response to monovalent
versus bivalent binding of the receptor. Monovalent binding was preferentially
associated with transcytosis, while bivalent binding preferentially led to
lysosomal compartmentalization," said David Hilbert, CSO of cancer antibody
company Zyngenia Inc. "This
will have important consequences for the further development of brain shuttles."

Richard Daneman, a fellow at the University of California, San Francisco,
added, "One of the nice things in the new monovalent configuration is that
it can be used easily with other therapeutic antibodies. It represents more of
a platform than other Trojan horse antibodies."

The reason, he said, is that the new Brain Shuttle
technology incorporates therapeutic antibodies "with their native
architecture."

"In principle, such an efficient brain shuttle can
revolutionize the development of medication for neurological disorders,"
said Bart De Strooper, a professor at the Catholic University Leuven
and head of the Department of Molecular and Developmental Genetics at the Flanders Institute for Biotechnology (VIB).

Shuttle service

Roche is already testing its Brain Shuttle
technology in combination with at least two therapeutic modalities-antisense
and antibodies.

For example, anti-TfR antibodies are associated with
defects in reticulocytes, immature red blood cells that express high levels of
the receptor required to take up transferrin-bound iron needed for hemoglobin
loading and cell maturation.5

Earlier this week, the Genentech team published data
showing how high-affinity, TfR-directed antibodies such as its TfR-BACE1
bispecific, chimeric antibody drive TfR into the lysosome for degradation. Over
time, TfR levels become depleted, rendering the pathway unavailable for
transcytosis of endogenous molecules and biologics.6

In contrast, Roche's monovalent TfR format could avoid
toxicity because it preserved normal TfR levels on the luminal side of the BBB
and did not induce TfR degradation in mice.

Michael Ehlers, SVP and CSO of the neuroscience research
unit at Pfizer Inc., wanted to see
more characterization of Brain Shuttle. "So far the distribution of the
antibody has only been assessed by immunofluorescence-based signal
amplification," he said. "It will be important to assess the antibody
fraction that reaches the brain parenchyma more directly at higher resolution
in time course experiments and to investigate where and how long it is
detectable."

Hilbert thinks that affinity and valency are "two
sides of the same coin" and that further work will help to define the role
of each in transcytosis. It would be interesting to test Brain Shuttle and Genentech's
approach head to head. "True affinities of the Trojan horse antibodies by
Genentech and Roche still need to be measured by more sensitive means such as
surface plasmon resonance," he said.

"It would make sense and be straightforward to compare
the different TfR-based Trojan horse shuttles side by side and quantify their
effective brain concentrations. In vivo assays could be used to further
translate such results," added Ehlers.

Moreover, he said that "more experiments should be
done in wild-type animals since mouse models of AD often have impaired vascular
integrity." Indeed, Ab
forms aggregates in the cerebrovasculature that can locally disrupt the BBB.

Deciphering the human BBB

Freskgård said that Roche is now pushing
ahead with plans to generate Brain Shuttle modules with primate and human TfRs.

Daneman said that primate- or human-specific anti-TfR
antibodies will likely have varying binding affinities and TfR functions.

"The BBB and the functions of individual transport
systems like TfRs likely differ between rodents and primates so that efficacy
seen in rodent models cannot necessarily be anticipated in humans. Therefore, a
more detailed understanding about the BBB in primates will be very useful,"
said Ehlers.

He said that a team at the University of Wisconsin-Madison
is developing human induced pluripotent stem (iPS) cell protocols that generate
in vitro systems able to recapitulate aspects of the human BBB.7

Roche's pRED has filed a patent application covering BBB
transport using the monovalent Brain Shuttle module. The IP is not available
for licensing.

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